Cationic amphiphiles

ABSTRACT

A cationic lipid useful in transfection of cells with DNA is disclosed.

RELATED CASE

This is a division of application Ser. No. 08/189,594, filed Jan. 31,1994, now abandoned, which is in turn a continuation-in-part ofapplication Ser. No. 07/751,873, filed Aug. 28, 1991, now U.S. Pat. No.5,283,185.

BACKGROUND OF THE INVENTION

The present invention relates to methods for facilitating the transferof nucleic acids into cells and to a novel cationic amphiphile usefulfor this purpose.

Some but not all cationic amphiphiles are known to facilitate thetransfer of DNA into cells, i.e., transfection. Although the mechanismof this activity is not yet clear, it probably involves the binding ofthe DNA/lipid complex with the cell surface via the excess positivecharges on the complex. Cell surface bound complex is probablyinternalized and the DNA is released into the cytoplasm of the cell froman endocytic compartment. How the released DNA moves into the nucleus isnot known.

A cationic amphiphile contains the following four important structuralelements:

lipophilic group, - - - Linker bond, - - - Spacer arm, - - - Amino group

The amino group is positively charged at neutral pH. It may be aprimary, secondary, tertiary or quaternary ammonium group. The spacerarm is usually a hydrophilic, 2 to 15-atom moiety which connects theamino group to the lipophilic group via the linker bond. The linker bondis either an ether, ester, amide or other hydrolyzable bond.

The lipophilic group is a hydrophobic moiety which allows the insertionof the cationic amphiphile into the membranes of the cell or liposome.It serves as an anchor for the cationic ammonium group to attach to thesurface of a cell or liposome.

N-[1-(2,3-dioleoxyloxy)propyl]-N,N,N-trimethyl ammonium chloride (DOTMA)is the first cationic amphiphile exhibiting the activity oftransfection. Its lipophilic group is a double-chain, C18:1 aliphaticgroup. It contains a quaternary ammonium group connected to thelipophilic group via a 3-carbon spacer arm with two ether linker bonds.Although the molecule is effective in transfection, it is notbiodegradable and is rather toxic to cells.

Another series of cationic amphiphiles used in transfection on is thequaternary ammonium detergents. Either single chain (such ascetyltrimethylammonium bromide) or double chain (such asdimethyldioctadecylammonium bromide) detergents exhibit activity totransfect animal cells. The amino group in these amphiphiles isquaternary and is connected to the lipophilic group without the spacerarm or linker bonds. Another single-chain detergent, stearylamine,contains a primary amino group connected to a single C18:0 chain withouta spacer arm or linker bond. This group of amphiphiles is also toxic tothe cells.

Two other groups of cationic amphiphiles for transfection have beenreported. The first group contains two C18:1 chains as the lipophilicgroup. The second group contains a cholesterol moiety as the lipophilicgroup. Both groups contain a quaternary ammonium group, but the spacerarm structure varies. In one case, the trimethylammonium group isdirectly connected to the two C18:1 chains via a 3-carbon spacer arm andester bond. The amphiphile, 1,2-dioleoxy-3-(trimethylammonio)propane,(DOTAP) is a close analog of DOTMA. In other cases, such as1,2-dioleoyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol, DOBT, orcholesteryl(4′-trimethylammonio)butonate, ChOTB, the trimethyl-ammoniumgroup is connected via a butanoyl spacer arm to either the double-chain(for DOTB) or cholesteryl (for ChOTB) group. Other amphiphiles, i.e.,1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC) and cholesterylhemisuccinate choline ester, ChOSC, contain a choline moiety as thequaternary ammonium group which is connected to the double-chain (forDOSC) or cholesteryl (for ChOSC) group via a succinyl spacer arm. Thetransfection activities of these amphiphiles are generally weak.

Yet another class of amphiphiles, called “lipopolyamine” has also beenreported. The ammonium group is L-5-carboxyspermine which contains 2primary and 2 secondary ammonium groups. Two examples of thislipopolyamine are dioctadecyl amidologlycylspermine, DOGS, anddipalmitoyl phosphatidylethanolamidospermine, DPPES. The cationic groupis connected to two different double-chain, C16:0 lipophilic group viaan amidoglycyl (for DOGS) or phosphorylethanolamine (for DPPES) spacerarm. These compounds are especially efficient in transfecting theprimary endocrine cells without cellular toxicity.

A lipopolylysine reagent for transfection has also been reported. Thereagent contains a polylysine moiety as the ammonium group which isconnected to a phospholipid (N-glutarylphosphatidylethanolamine).Therefore, the spacer arm is the side chain of lysine and the head groupof the phospholipid. The lipophilic group is a double-chain, C18:1 groupconnecting to the spacer arm via two ester bonds. Although the reagentis efficient in transfection and non-toxic to cells, the activityrequires scraping the treated cells. This is clearly not a convenientstep and cannot be done for in vivo experiments.

An ideal transfection reagent should exhibit a high level oftransfection activity without scraping or any other mechanical orphysical manipulations of the cells or tissues. The reagent should benon-toxic or minimally toxic at the effective doses. It should also bebiodegradable to avoid any long-term adverse side-effects on the treatedcells.

Many reagents which fulfill these criteria contain a linker bond that ishydrolyzable in the cell. For example, DOBT and DOSC, both contain esterlinker bonds, can be metabolized and catabolized into other lipidspecies in the treated cells. However, cationic amphiphiles containingester linker bonds are not stable when stored in an aqueous solution.This is probably due to a base-catalyzed hydrolysis reaction mediated bythe amino group of the amphiphile.

Another key factor on the cellular toxicity of the cationic amphiphilesis their inhibitory effects on the activity of protein kinase C (PKC).PKC is a key enzyme which plays a crucial role in cellular signaltransduction. Cationic amphiphiles inhibit PKC activity by mimicking theendogenous inhibitor, sphingosine. PKC activity is also important forthe cellular endocytosis pathway which is likely to be involved in theaction of the cationic amphiphiles to facilitate the entry of DNA intocells. Recently it has been reported that a PKC activator,phorbolmyristateacetate, can stimulate the transfection efficiency ofDNA mediated by the calcium phosphate precipitates.

SUMMARY OF THE INVENTION

The present inventors have therefore synthesized a series of novelcationic amphiphiles and screened their activities to inhibit PKC.Several amphiphiles which exhibit weak inhibitory activities towards PKCare particularly suitable for transfections. In addition, there has beenprepared cationic reagents with a carbamoyl linker bond in order toovercome the problem of instability in solution. The stability of thebond in aqueous solution is much greater than that of the ester bond,yet it is hydrolyzable in the cell.

In brief, the present invention provides a method for facilitating thetransfer of nucleic acids into cells. The method comprises preparing amixed lipid dispersion of a cationic lipid with a co-lipid in a suitablecarrier solvent, such as distilled water or normal saline solution. Thecationic lipid has a structure which includes a lipophilic group derivedfrom cholesterol, a linker bond, a spacer arm including a moiety of 1 toabout 20 atoms, usually alkyl of 1 to 6 carbon atoms, in a branched orunbranched linear alkyl chain, and a cationic amino group. The aminogroup is selected from the group consisting of primary, secondary,tertiary and quaternary amino groups. The method further comprisesadding the nucleic acids to the dispersion to form a complex. The cellsare then treated with the complex.

In a preferred embodiment of the invention, the dispersion has particleswith an average diameter of about 150nm. The cationic lipid ispreferentially selected from the group consisting ofcholesteryl-3β-carboxyl-amidoethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylateiodide, cholesteryl-3β-carboxyamidoethyleneamine,cholesteryl-3β-oxysuccinamidoethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3β-oxysuccinateiodide,2-[(2-trimethlyammonio)-ethylmethylamino]ethyl-cholesteryl-3β-oxysuccinateiodide, 3β[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol, and3β-[N-(polyethyleneimine)-carbamoyl]cholesterol.

In a preferred embodiment, the co-lipid is a neutral or acidicphospholipid which may be preferentially selected from the groupconsisting of phosphatidyl choline and phosphatidyl ethanolamine.

In addition, the present invention also provides a substantiallynon-toxic,substantially non-hydrolyzable cationic lipid for facilitatingthe transfer of nucleic acids into cells. The lipid comprises alipophilic group derived from cholesterol, a linker bond, a spacer armincluding from about 1 to about 20 carbon atoms, preferably 1 to 6carbon atoms in a branched or unbranched linear alkyl chain, and acationic amino group. The amino group is selected from the groupcomprising primary, secondary, tertiary or quaternary amino groups.

The cationic lipid is preferably selected from the group consisting ofcholesteryl-3β-carboxyamidoethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylateiodide, cholesteryl-3β-carboxyamidoethyleneamine,cholesteryl-3β-oxysuccinamidoethylenetrimethylammonium iodide,1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3β-oxysuccinateiodide,2-[(2-trimethyl-ammonio)-ethylmethylamino]ethyl-cholesteryl-3β-oxysuccinateiodide,3β[N-(N′,N′dimethylaminoethane)-carbamoyl]-cholesterol, and3β[N-(polyethyleneimine)-carbamoyl]cholesterol.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention may be better understood by reference to thefollowing Examples when considered in conjunction with the drawings inwhich:

FIG. 1 is the synthetic scheme for cholesteryl carboxylate analogues;

FIG. 2 is the synthetic scheme for cholesteryl hemisuccinate analogues;

FIG. 3 is the synthetic scheme for cholesteryl formate analogues;

FIG. 4 is a graph of the effect of different co-lipids on thetransfection activity of a cationic lipid dispersion in L929 cells;

FIG. 5 is a graph of the effect of the ratio of co-lipid to a cationiclipid of the present invention on the transfection activity in L92.9cells;

FIG. 6 is a graph of the effect of lipid dose on the transfectionactivity in L929 cells;

FIG. 7 is a graph of the effect of DNA dose on the transfection activityof the lipid dispersion in L929 cells;

FIG. 8 is a representation of a gel showing complex formation of DNAwith the cationic lipid dispersion; and

FIG. 9 is a graph of the transfection efficiency and toxicity of acationic lipid of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

When used in gene therapy, the dispersions of the invention containingat least one cationic lipid of the invention may be used to deliver DNAinto the selected eukaryotic cell. Protocols for stable transformationand expression of DNA integrated into the genome of the transfected cellare known. Typical protocols for liposome-mediated transfections aredescribed in Ausebel et al. Current Protocols in Molecular Biology,Volume 1, Unit 9.4.1 and, also generaly, see Chapter 9 for Introductionof DNA into Mammalian Cells.

The dispersions of the invention can also be used to introduce nucleicacid, e.g. plasmid DNA into protoplasts of prokaryotic cells by methodsknown in the art.

The dispersions of the invention can be used to introduce nucleic acidsinto protoplasts of plant cells. Phospho-lipids vesicles have been usedfor intracellular delivery of liposomal contents into plant cells inreported work with tobacco protoplasts. Tobacco mosaic virus (TMV), RNAhas been encapsulated in liposome preparations using the reverseevaporation method developed by Szoka and Papahadjopoulos. See PNAS USA75:4194-4198 (1978). Studies with a variety of plant species (flower andvegetable), like tomato, lily, daylily, onion, peas, petunia and othershave been reported. See, Genetic Engineering of Plants, Ed. Kosuge,Merideith and Hollaender, published by Plenum Press, authored by Fraleyand Horsch, entitled “In vitro Plant Transformation Systems UsingLiposomes and Bacterial Co-Cultivation”, Vol. 26, pps. 177-194 (1983)and other articles therein, which are incorporated herein by reference.Phosphatidyl serine-cholesterol (PS-Chol) (an anionic liposome), andother liposomes with encapsulated RNA have been reported. See Fraley etal (above cited). The protocols are reported to be useful to introduceRNA and/or, DNA molecules into the plant protoplasts. In a similarmanner, the dispersions of the invention with appropriate adaptation byone skilled in the art to best fit the purpose intended, can be used totransform plants. A cationic lipid of particular interest is3β[N-(N′,N′-dimethyl-aminoethane)-carbamoyl]cholesterol.

A dispersion of the invention containing3β[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol and plasmid DNAis suitable for direct injection into the tumor lesion of a patient.Such a dispersion can be applied as an aerosol into the airways, such asthe trachea, the nasal or other cavities of a cystic fibrosis patient.Likewise, such a dispersion may be contemplated for peritonitalinjection into a patient with ovarian carcinoma with metastasis in theperitonital cavity. For the treatment of neurological diseases likeAlzheimer disease, direct injection and transfection of brain cells tocause expression of a therapeutic copy of the defective target gene isof major interest. The dispersions of the invention are likewiseconsidered useful for gene therapy of muscular dystrophy, hemophilia Band several other diseases caused by defective genes.

Instead of a dispersion containing the cationic lipid identified above,the dispersion may contain one or more of the cationic lipids of theinvention. It is not excluded to use other cationic lipids with one ormore cationic lipids of the invention, providing the formulation isadequately stable and effective for cell transfection. One skilled inthe art with the knowledge of the properties of the cationic lipids ofthe invention (and with the knowledge of the other lipids) can readilyformulate a dispersion best suited for the particular cell transfectiondesired.

In order to facilitate a further understanding of the present invention,the following Examples are given primarily for the purposes ofillustrating certain more specific details thereof.

MATERIALS

Cholesterol (99+% grade), cholesterol hemisuccinate,1,1′-carbonyldimidazole, were purchased from Sigma Chemical Co., St.Louis, Mo. Magnesium powder-50 mesh (99+%), thionyl-bromide (97%),1,3-propane sulfone (99%), iodomethane (99%), trans-1,2-dichloroethylene(98%), M,M-dimethylaniline (99%), N,N-dimethylethylenediamine (95%),1,3-bis-dimethylamino-2-propanol (97%),2-{[2-(dimethylamino)ethyl]methylamino}ethanol (98%), were obtained fromAldrich Chemical Co., Milwaukee, Wis. Cholesteryl chloroformate (95%),and polyethyleneimine were obtained from Fluka. Methanol,dichloromethane, and acetonitrile were HPLC grade solvents. All otherchemicals and solvents, unless specified were reagent grade.

A synthetic scheme for cholesteryl carboxylate analogues is shown inFIG. 1.

EXAMPLE 1 Cholesteryl Bromide (I)

Cholesterol, (25 g, 64.6 mmol) was dissolved in 10 ml of dimethylaniline(78.9 mmol) and 5 ml of chloroform. While stirring on ice; smallquantities of thionyl bromide (6 ml, 77.6 mmol) dissolved in 20 ml ofcold chloroform was added slowly over a period of 15 minutes. After theaddition of thionyl bromide was complete, the mixture was stirred for anadditional 2 hours at room temperature. The resulting solution waspoured into 200 ml of ice cold 95% ethanol and left on ice for 2 houruntil crystallization was complete. The product was filtered and washedwith 25 ml of ice cold 95% ethanol. A small amount of product wasrecovered from the filtrate with the addition of 75 ml distilled waterfollowed by refrigeration. Finally, the product was recrystallized from120 ml of acetone giving 21.8 g of cholesteryl bromide (yield, 75%) witha melting point of 93-95% C. (lit 97-98° C.). The identity of theproduct was confirmed with mass spectrometry (EI) which showed anintense peak with an m/z of 448, corresponding to the molecular ion(M⁺°) of cholesteryl bromide. Also, the bromide molecular weight patterncharacteristic of the two different isotopes of bromine (79Br:81Br,1:1)was observed.

EXAMPLE II Cholest-5-ene-3β-Carboxylic Acid (II)

The synthesis of cholesteryl-3β-carboxylate was performed using aGrignard reaction. All glassware was oven dried at 110° C. overnight. Ina 500 ml three-neck flask set up for reflux, a solution of methylmagnesium iodide was freshly prepared by treating 9 g of oven dried(110° C.) magnesium powder in 100 ml anhydrous diethyl ether with 10 mlof methyl iodide. After the vigorous reaction subsided, cholesterylbromide (25 g, 56 mmol) dissolved in 100 ml of anhydrous diethyl etherwas slowly added to the methyl magnesium iodide solution over a threehour period. The solution was refluxed for 36 hours with enough heatrequired to bring the diethyl ether to a boil. Subsequent to cooling,the Grignard reagent was added to finely ground solid carbon dioxide,and after 1 hour, the complex was hydrolyzed by treatment with ice cold1 M sulfuric acid. After the steroid was extracted with diethyl ether(3×250 ml), the ethereal layer was washed with 10 mM sodium thiosulfate(3×50 ml) to remove a persistent orange color. After removing the waterlayer, the ether layer was washed with distilled water and filtered toremove an insoluble residue. The ether layer was subsequently dried overanhydrous sodium sulfate and rotary evaporated to give a white-yellowoily suspension. Titration with pentane yielded 8.6 g ofcholesteryl-3β-carboxylate (yield, 37%) as a fine powder with a meltingpoint of 212-215° C. (lit 218-220° C.). Mass spectrometry (EI) showed anm/z of 414 of the molecular ion (M+°). The product was characterized by21 proton NMR. The product was lyophilized overnight to give ananhydrous starting material for acylation reactions.

EXAMPLE III Cholesteryl-3β-Carboxyamidoethylenedimethylamine (III)

The acylation of cholesteryl carboxylate was carried out under a dryargon or nitrogen atmosphere in oven dried glassware. Cholesterolcarboxylate (2 g, 4.8 mmol) was suspended in 5 ml of dichloromethane(HPLC grade under 4 Å molecule sieves). A 1.5 molar excess of1,1′-carbonyldimidazole (CDI, 1.2 g) dissolved in 15 ml dichloromethanewas added to the cholesteryl carboxylate suspension is small volumeswith intermittent shaking. When the reaction subsided, the solution wasstirred overnight. N,N-dimethylethylenediamine (5 ml, 43.2 mol) wassubsequently added and the resulting solution was stirred for 36 hoursat room temperature. Dichloromethane was removed by rotary evaporation,after which the reaction was quenched with a small volume of distilledwater. The acylated steroid was extracted with diethylether (4×50 ml).Subsequently, the pooled ether fractions were back extracted withdistilled water (3×50 ml), dried over anhydrous sodium sulfate, androtary evaporated under reduced pressure. The residue was thentriturated with pentane and the product collected on a sintered glassfunnel. A voluminous powder (1.7 g, 73% yield) was obtained and found tobe pure by TLC (Rf=0.72) using chloroform:methanol:water (65:25:4,v/v/v)as the developing solvent. The product gave a melting point of 167-169°C. Mass spectrometry (FAB+) showed an intense peak at an m/z of 485which corresponds to the protonated molecular ion (M+H)⁺°. The productwas characterized by proton NMR.

EXAMPLE IV Cholesteryl-3β-Carboxyamidoethylenetrimethylammonium Iodide(IV)

The quaternization of Compound III was performed using methyl iodide andpotassium bicarbonate. Briefly, 1 g (2.1 mmol) of compound III wasdissolved in 40 ml of methanol in the presence of 2 g (20 mmol) ofpotassium bicarbonate and 2 ml (32.1 mmol) of methyl iodide. Thereaction was stirred for 24 hours at room temperature. The solvent wassubsequently removed under vacuum and the remaining bicarbonate wasneutralized with 1 M HCl until the solution gave a pH reading of 7.Water was removed by lyophilization and the product was extracted frominorganic salt impurities using a small volume of ice cold methanol.After evaporating the solvent, the product was recrystallized fromabsolute ethanol and was further purified on a reverse phase columnusing an acetonitrile/0.1% trifluoroacetic acid gradient (100% to 85%acetonitrile in 60 minutes). The powder was shown to be pure with TLC(Rf=0.10) using chloroform:methanol:water (65:25:4 v/v/v) as thedeveloping solvent. It was shown to melt with decomposition at about190° C., and had a molecular ion with an m/z of 500 (M⁺°) according tomass spectrometry (FAB+). The product was characterized by proton NMR.

EXAMPLE V 1,3-Bis-Dimethylamino-2-Propyl-Cholesteryl-3β-Carboxylate (V)

Acylation was performed using CDI activated cholesteryl-3β-carboxylateanalogous to the method described for compound III, except that2,3-bis-dimethylamino-2-propanol (8 ml, 47.6 mmol) was the nucleophile.After the addition of the nucleophile, the reaction was stirred at roomtemperature for 72 hours. The dichloromethane was removed and theremaining oily residue was dissolved in chloroform. Impuritiesprecipitated with a large volume of petroleum ether (bp, 35-60° C.). Thefiltrate was rotary evaporated to dryness, re-dissolved in pentane, andfiltered once again. After drying, the pentane soluble material wasdried and re-dissolved in a small volume of diethyl ether and added to alarge volume of hot diethyl ether:acetonitrile (30:70, v/v). The productcrystallized at −20° C. after allowing some of the ether to evaporate.Mass spectroscopy (FAB+) gave an m/z of 543 for the protonated molecularion (M+H)+°. The product was characterized by proton NMR.

EXAMPLE VI 1-Dimethylamino-3-Trimethylammonio-DL-2-Propyl CholesterylCarboxylate Iodide Salt (VI)

The methoidide of compound V was prepared by gently refluxing compound V(0.5 g, 0.9 mmol) and methyl iodide (2 ml, 32.1 mmol) in 20 ml ofethanol for one hour. After cooling, the precipitate (0.5 g, yield 79%)was recrystallized twice from absolute methanol. The product melted withdecomposition at about 232° C. and ran as a single spot on a TLC plate(Rf=0.22) using chloroform:methanol:water (65:25:4, v/v/v) as thedeveloping solvent. The product had a molecular ion with an m/z of 557(M⁺°) with FAB+ mass spectroscopy, consistent with the alkylation of oneof the possible two tertiary amine sites. The product was characterizedby proton NMR.

EXAMPLE VII Cholesteryl-3β-Carboxyamidoethyleneamine (VII)

To a solution of ethylenediamine (5.11 g, 85 mmol) in 20 mldichloromethane, a solution of CDI activated cholesteryl carboxylate(0.7 g, 1.7 mmol) in 5 ml of dichloromethane was added dropwise over a1.5 hour period. When the addition of the activated sterol was complete,the~reaction was stirred for 48 hours under nitrogen. After removing thesolvent under reduced pressure, the residue was dissolved inchloroform:methanol (2:1, v/v) and extracted against water (3×50 ml).The chloroform phase was subsequently dried with anhydrous sodiumsulfate, the solvent removed and the residue purified by preparative TLCusing chloroform:methanol:water (65:25:4, v/v/v) as the developingsolvent. The band at about Rf=0.3 was collected, extracted withchloroform:methanol (1:1, v/v) and dried under reduced pressure. Theproduct (0.65 g, yield, 81%) ran as a single spot (Rf=0.33) and meltedwith decomposition at about 194° C. Mass spectrometry (FAB+) gave an m/zof 457 for the protonated molecular ion (M+H)+. The product wascharacterized by proton NMR.

A scheme describing the various steps for producing cholesterolhemisuccinate analogues is depicted in FIG. 2.

EXAMPLE VIII Cholesteryl-3β-Oxysuccinamidoethylenedimethylamine VIII

The synthesis of compound VIII first required the acyl imidazoline ofcholesteryl hemisuccinate which was prepared by reacting cholesterolhemisuccinate with N,N-carbonyldiimidazole (CDI) as described for thesynthesis of compound III. Briefly, to cholesterol hemisuccinate (2 g,4.1 mmol) suspended in 5 ml of dichloromethane was added 1.5 equivalentsof CDI (1 g) dissolved in 15 ml of dichloromethane. The solution wasstirred overnight after which N,N-dimethylethylenediamine (5 ml, 43.2mmol) was added. Dichloromethane was subsequently removed by rotaryevaporation, distilled water was added and the acylated sterol wasextracted with diethyl ether (4×50 ml). Subsequently, the etherfractions were washed with distilled water (3×50 ml) and dried overanhydrous sodium sulfate. The ether was removed by rotary evaporation.The product was washed with 200 ml of pentane, and minor impurities wereremoved using preparative silica gel TLC. After developing withchloroform:methanol:water (65:25:4), v/v/v) the band present at about anRf=80 was collected and extracted with chloroform/methanol (2:1 v/v).The residue was purified further using chloroform:ethyl acetate (1:1,v/v) as the second developing solvent. The band at about Rf=0.2 wasextracted with chloroform/methanol (2:1 v/v). The lyophilized productran as a single spot on TLC with an Rf of 0.75 usingchloroform:methanol:water (65:25:4, v/v/v) as the developing solvent andhad a melting point of 119-111° C. Mass spectrometry (FAB+) showed anm/z of 557 which would correspond to the protonated molecular ion(M+H)^(+°). The product was characterized by proton NMR.

EXAMPLE IX Cholesteryl-3β-Oxysuccinamidoethylenetrimethylammonium Iodide(IX)

The quaternization of compound VIII was carried out with methyl iodidein absolute ethanol as described earlier for the synthesis of compoundVI. Allowing the solution to cool to room temperature afforded 0.5 g(80% yield) of the quaternary ammonium salt. Subsequently, the productwas recrystallized from absolute ethanol giving a fine white powderwhich melted with decomposition at about 196° C. The product ran as asingle spot on a TLC plate (R=0.43) using chloroform:methanol:water(65:25:4) as the developing solvent. Mass spectrometry (FA+) indicated amolecular ion with an m/z of 572 (M⁺°). The product was characterized byproton NMR.

EXAMPLE X 1,3-Bi-Dimethylamino-2-Propyl-Cholesteryl-3β-Oxysuccinate (X)

Acylation was performed using CDI activated cholesteryl hemisuccinateaccording to the procedure described earlier for compound V. After theaddition of 1,3-bi-dimethylamino-2-propanol (7 ml, 41.6 mmol), themixture was stirred for 72 hours, after which the solvent was removedunder vacuum. The product, extracted from the residue with diethyl ether(3×75 ml), gave an oil following removal of the ether. The addition ofpentane precipitated additional impurities; after rotary evaporation,the resulting oil could not be successfully crystallized using a varietyof solvents or by lyophilization. Mass spectrometry (FA+) indicated aprotonated molecular ion with an m/z of 616 (M+H)⁺°. the product wascharacterized by proton NMR.

EXAMPLE XI1-Dimethylamino-3-Trimethylammonio-DL-2-Propyl-Cholesteryl-3β-OxysuccinateIodide Salt (XI

Methylation of compound X was performed using the method describedpreviously (Example VI). After 1 hour, the solution was cooled and themethodide recrystallized twice from absolute methanol to give needleshaped crystals which melted with decomposition at about 222° C. Theproduct ran as a single spot on a TLC plate (R=0.17) usingchloroform:methanol:water (65:25:4, v/v/v) as the developing solvent.Mass spectrometry (FA+) indicated a molecular ion with an m/z of 629(M⁺°) consistent with the methylation of 1 of a possible 2 tertiaryamine sites. The product was characterized by proton NMR.

EXAMPLE XII2-{[2-(dimethylamino)ethyl]methylamino}ethyl-Cholesteryl-3β-Oxysuccinate(XII)

The synthesis of compound XII was analogous to the method described forthe acylation of compound VIII except that2-{[2-dimethylamino)ethyl]-methylamino}ethanol (7 ml, 42.0 mmol) was theamino alcohol used as the nucleophile. After extraction withdiethylether, the product was lyophilized dry and further purified bypreparative TLC using chloroform:methanol:water (65:25:4, v/v/v). Afterthe band present at R=0.80 was collected and extracted withchloroform:methanol, (2:1, v/v), the residue was purified further usingchloroform:ethyl acetate (1:1; v/v) as the second TLC developingsolvent. The band which was present at about Rf=0.2 was collected andthe silica was extracted with chloroform:methanol (2:1, v/v). Theproduct, which ran as a single spot on a TLC plate (Rf=0.72) usingchloroform:methanol:water (65:25:4 v/v/v) as the developing solvent gavea melting point of 50-52° C. Mass spectrometry (FA+) showed a protonatedmolecular ion with an m/z of 615 (M+H)⁺°. The product was characterizedby proton NMR.

EXAMPLE XIII2-{[2-Trimethylammonio]ethylmethylamino}ethyl-Cholesteryl-3β-OxysuccinateIodine Salt (XIII)

The acylation of compound XII (0.5 g, 0.8 mmol) was carried out underreflux conditions with methyl iodide in absolute ethanol as described inExample VI. The precipitate was recrystallized twice from absolutemethanol and stained as a single spot on a TLC plate (Rf=0.22) usingchloroform:methanol:water (65:25:4; v/v/v) as the developing solvent.The crystals melted with decomposition at about 172° C. Massspectrometry (FA+) gave an m/z of 629 for the molecular ion (M⁺°)consistent with the methylation of only one the possible two tertiaryamine sites. The product was characterized by proton NMR.

The scheme for the synthesis of cholesteryl formate analogues is shownin FIG. 3.

EXAMPLE XIV 3β[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (XIV)

Compound XIV was synthesized by mixing a solution of cholesterylchloroformate (0.5 mmol) in chloroform with a solution ofN,N-dimethylethylenediamine (9.1 mmol) in chloroform in a dryice-ethanol bath. The solvent and the unreacted amine were removed invacuo. Compound XIV was purified by two successive recrystallizations inethanol. (Yield, 65%) TLC (chloroform:methanol=65:35) showed a singlespot (Rf=0.37) when developed with iodine. The product was characterizedby proton NMR.

EXAMPLE XV 3β[N-(polyethyleneimine)-carbamoyl]cholesterol XV)

Synthesis of compound XV was similar to that of compound XIV.Cholesterol chloroformate (0.1 mmol) and polyethyleneimine 600 (6 g)were mixed in chloroform in a dry ice-ethanol bath. After the volatilematerial of the reaction mixture was removed in vacuo, the solid crudeproduct was dialyzed against 4L distilled water for 3 days (during whichthe water was changed several times). Finally, the product waslyonhilized to dryness, giving an estimated yield of 81%. Compound XVran as a single spot on TLC (chloroform:methanol=65:35).

EXAMPLE XVI Preparation of Cationic Livid Dispersions

The cationic cholesterol derivatives of the invention were mixed with aphospholipid co-lipid in chloroform solution at different molar ratios.The solvent was removed by evaporation under a stream of N₂ gas anddesiccated in vacuo for at least 30 minutes. The dry lipid film washydrated in 20 mM Hepes buffer, pH 7.8, overnight. The suspension wassonicated in a bath-type sonicator (Laboratory Supplies, Hicksville,N.Y.) to generate small particle dispersions (average diameter=150 nm).The co-lipid is phosphatidylethanolamine.

EXAMPLE XVII Transfection of Cells

Plasmid pUCSV2CAT (approximately 5 kb in size) containing the structuralgene of E. coli chloramphenicol acetyl transferase (CAT) driven by theSV40 virus early promoter was used as a model for the polyanions to bedelivered by the cationic lipid dispersions. DNA was mixed with thecationic lipid dispersions of the invention in 1 ml serum-free M199medium or McCoy's medium to form DNA/lipid complex. Cultured mammaliancells of about 80-100% confluency in a 6-well plate were washed oncewith serum-free medium. The DNA/lipid complex was added to the washedcells which were incubated at 37° C. for 5 hours. The cells were washedagain and the serum-containing medium was added. Cells were harvested30-72 hours later and extracted for cellular proteins. The CAT activityin the extracted protein was measured by using either [¹⁴C]chloramphenicol or [³H] acetyl CoA as a radiolabeled substrate. Oneactivity unit of CAT is defined as nmole of radiolabeled substrateconverted to the radiolabeled product in one minute. Protein content inthe cell extracts was measured by the Bradford (BIORAD) assay.

EXAMPLE XVIII Isolation of Protein Kinase C

As rapidly as possible, brains for 25 Sprague-Dawley rats (150-200 g)were removed, washed with 100 ml of 20 mM TRIS, 1 mM EDTA, 1 mM EGTA, pH7.5, and homogenized in 150 ml of ice cold 20 mM TRIS, 10 mM EGTA, 2 mMEDTA, 10 mM DTT, 0.25 M sucrose, 2 mM PMSF and 100 μg/ml leupeptin, pH7.5. The homogenate was immediately centrifuged at.100,000 g for 40minutes at 4° C. in a Beckman Ti 50.2 rotor. The supernatant was appliedto a 2.5×20 cm column of DEAE Sepharose (fast flow) containing 60 ml ofresin equilibrated with 20 mM TRIS, 1 mM EDTA, 1 mM DTT, pH 7.5 (bufferA). The column was washed with 300 ml of buffer A and an additional 200ml of buffer A containing 0.03 M KCl. Protein kinase C was eluted with a500 ml continuous KCl gradient (0.03-0.3 M KCl). Fractions of 5 mlvolumes were collected. Fractions showing calcium and phospholipiddependence were pooled; the salt concentration was adjusted to 1.5 KClwith the appropriate quantity of solid KCl. The crude sample containing1.5 M KCl was stirred for 15 minutes and subsequently loaded onto a 1×10cm column containing 9 ml Phenyl sepharose equilibrated with 1.5 M KClin 20 mM TRIS, 0.5 mM EGTA, 1 mM DTT, pH 7.5 (buffer B). The column waswashed with 90 ml of buffer B containing 1.5 M KCl. PKC was eluted witha 100 ml continuous KCl gradient (1.5-0 M KCl). Fractions of 3 mlvolumes were collected. The column was washed with an additional 50 mlof buffer B. Most of the enzyme activity eluted during this stage.Fractions showing calcium and phospholipid dependence were pooled andconcentrated to 4 ml using an Amicon ultrafiltration cell fitted with aYM-10 filter. The concentrated sample was loaded onto a 2.5×100 cmcolumn containing 400 ml of Sephacryl S-200 HR beads equilibrated withbuffer B containing 10% glycerol (buffer C). Fractions of 3 ml volumeswere collected. About 150 ml of buffer was run through; PKC eluted veryclose to the column void volume. The fractions showing calcium andphospholipid dependence were pooled and loaded onto a 0.5×5 cm columncontaining 2.5 ml polylysine agarose equilibrated with buffer C. PKC waseluted with a 40 ml continuous KCl gradient (0-0.8 M KCl). Fractions of1 ml volumes were collected. The first few active fractions werecontaminated. The uncontaminated fractions were pooled, concentrated,and diluted with buffer C to remove the high salt content. Afterreconcentrating, the sample was divided into working portions, frozen inliquid nitrogen and stored at −80° C. Full activity was regained afterrapid thawing. Trace impurities (116 k, 66 k, and 50 k Mr) could stillbe detected when the gel was silver stained heavily. The enzyme gave aspecific activity of 200 nmoles phosphate incorporated per minute permilligram of protein when assayed for histone phosphorylation using theTriton mixed micelle assay with 6.5 mole % phosphatidylserine, 2.5 mole% DAG and 100 μM calcium present. Specific activities ranging from 30nmoles/min/mg to 600 nmoles/min/mg have been observed for PKC using theTriton mixed micelle assay under the same conditions.

The DNA was mixed with the cationic lipid dispersion containing theco-lipid (DOPE) and3β[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol and transfectionis performed as shown in Example XXII.

Similar mixtures are obtained with other dispersions containing aselected co-lipid and other cationic lipids of the preferred groupdescribed above like cholesteryl-3β-carboxamidoethylenetrimethylammoniumiodide, cholesteryl-3β-carboxyamidoethylenamine,cholesteryl-3β-oxysuccinamidoethylenetrimethylammonium iodide, and3β-[N-(polyethyleneimine)-carbamoyl]cholesterol. Transfection of themammalian cells was performed as shown below in Example XXII or byprotocols referred to above.

EXAMPLE XIX Mixed Micelle Assay of Protein Kinase C

Phosphatidylserine and 1,2-diolefin with and without additive weredissolved in a solution of chloroform/methanol (2:1, v/v). Solvent wasevaporated with a stream of nitrogen and last traces removed using avacuum desiccator at 40° C. The lipid films were then solubilized by theaddition of 3% Triton X-100, vortexed vigorously for 30 seconds and thenincubated at 30° C. for 10 minutes to allow for equilibration. At 25 μl,an aliquot of this solution was used in a final assay volume of 250 μl,containing 20 mM TRIS-HCl, pH 7.5, 10 mM MgCL₂, 200 μg/ml histoneIII-S,100 μM CaCl₂, 10 μM[γ-³²P] adenosine 5′ triphosphate, 2.75 mM TritonX-100, with 300 μM (6.5 mole percent) phosphatidylserine and 107 μM (2.5mole percent 1,2-diolefin. For controls, 25 μl of 20 mM EGTA replacedthe CaCl₂. To initiate the reaction, 150 ng of protein was added. Afterbriefly mixing, the tubes were incubated for 10 minutes at 30° C. Thereaction was terminated by adding 1 ml of cold 0.5 mg/ml BSA and 1 ml ofcold 25% trichloroacetic acid. This mixture was passed through a GF/CWhatman filter and washed five times with 2 ml of 25% trichloroaceticacid. After drying, the filters were counted with 6 ml ACS scintillationfluid.

EXAMPLE XX Formation of Homogenous Dispersion with Cationic CholesterolDerivatives

None of the cationic cholesterol derivatives by themselves form stablehomogenous dispersion by sonication in a low ionic strength buffer. Whenstable dispersions are desired, it was necessary to add an acidic orneutral phospholipid to form mixed lipid dispersion. For example,compound VIII requires a minimal of 1 part of PC or PE and 9 parts ofcompound VIII to form a uniform dispersion. In the case of compound XIV,a minimal ration of phosphatidyl choline (PC) or phosphatidylethanolamine (PE) to XIV=4:6 is required. Such non-cationic lipid usedin the dispersion is called co-lipid.

EXAMPLE XXI Delivery of DNA into Mammalian Cells by Cationic LipidDispersions

Plasmid DNA, pUCSV2CAT, was used as a model compound for polyanionsbecause it contains a structural gene for CAT. The efficiency ofintracellular delivery can be readily assayed by the expression of CATactivity in the extracted proteins of the treated cells. Table 1 liststhe CAT activity of mouse L929 cells which have been transfected withthis plasmid DNA as mediated by various cationic lipid dispersions. Inaddition, the inhibitory activity of the pure cationic cholesterolderivatives on diolefin, phosphatidyl serine (PS), and Ca²⁺stimulatedprotein kinase C was also measured. This activity was expressed as anIC₅₀, which is the concentration at which 50% of PKC activity wasinhibited. As can be seen from Table I, derivatives giving low IC₅₀values, i.e., those strong PKC inhibitors, were not a good deliveryvehicle for DNA. For example, compounds IV, XI, VI and XIII, all havinga IC₅₀ value less than 20 μM, produced minimal CAT activities in thetreated cells. Among the ones which gave rise to high CAT activities,derivatives with a single tertiary amino group (compounds VIII, VI andIII) were more effective in delivering DNA than similar analogscontaining a single quaternary amino group (compounds IX and IV).Furthermore, among the derivatives with the same amino head group, thosecontaining a longer spacer arm (compounds VIII and IX) delivered agreater quantity of DNA than those containing a shorter spacer arm(compounds X, XI, V, VI and XV) were generally less effective deliveryvehicles.

Compound VII deserves some special attention. It contains only a singleprimary amino group with a short spacer arm, yet the transfectionactivity was relatively high.

TABLE I PKC Inhibition Relative CAT Compound IC₅₀ (μM) Activity III 25818 IV 12 0.7 V 643 2 VI 11 1 VII 246 68 VIII 191 100 IX 59 50 X 408 0.5XI 15 11 XII 164 19 XIII 20 14 XIV >1,000 75 XV — 11

EXAMPLE XXII The Importance of the Co-lipid

The experiments described in Example XXI were done with a lipiddispersion containing a cationic cholesterol derivative and a co-lipiddioleoyl phosphatidylethanolamine (DOPE). We have studied the role ofco-lipid in the delivery efficiency. FIG. 4 shows the data of anexperiment in which compound VIII was mixed with a variety of differentco-lipid, neutral and acidic, at a molar ratio of 1:1. The DNA deliveryactivity of these mixed dispersions were then studied. As can be seen,only DOPE supported the delivery activity of compound VIII. Otherneutral lipids such as dioleoyl phosphatidylcholine (DOPC),N-methyl-DOPE, N,N-dimethyl DOPE had little or no activity. None of theacidic lipids, such as PS and phosphatidylglycerol (PG) showed anyactivity.

The molar ratio of Dope and compound VIII in the dispersion also playedan important role. FIG. 5 shows that maximal DNA delivery activity ofthe dispersion occurred when the dispersion contained 20-50% compoundVIII. Too much or too little of compound VIII in the mixed dispersiondid not yield good delivery activity.

EXAMPLE XXIII Optimization of Dispersion-to-DNA Ratio for Delivery

A 1:1 mixture of compound VIII and DOPE were used to study the optimalratio of dispersion-to-DNA for delivery. FIG. 6 shows the data of anexperiment in which various amounts of dispersion were added to a fixedamount of DNA (5 μg) for transfection. Maximal activities occurred at69-80 nmoles of dispersion. We then used 70 nmoles dispersion and variedthe amount of DNA for transfection (FIG. 7). The bell-shaped curve inthe figure indicates that a 5 μg DNA gave the maximal activity. Thus theoptimal ratio of dispersion-to-DNA was 70 nmole lipid for 5 μg DNA.

EXAMPLE XXIV Complex Formation of DNA with Cationic Lipid Dispersions

It was expected that polyanions were complex with the cationic lipiddispersion via electrostatic interactions. Again, a 1:1 mixture ofcompound VIII and DOPE was used for the study. We have characterized thedispersion/DNA complexes by agarose gel electrophoresis. As shown inFIG. 8, 1 μg plasmid DNA electrophoresed as two closely located bands inthe gel (lane 1), which could be completely digested if DNAse wasincluded in the incubation buffer (lane 7). Incubation mixturescontaining increasing amounts of dispersion showed decreasingintensities of DNA bands (lanes 2, 3, 4, 5 and 6). Furthermore, all ofthe uncomplexed, free DNA could be digested by DNAse, but only a portionof the complexed DNA was digested (lanes 8, 9, 10, 11 and 12). Theseresults clearly showed that the lipid dispersion form complexes with DNAwhich are either larger in size and/or less negatively charged such thatthe complex does not enter the gel during electrophoresis. Furthermore,the complex is partially resistant to DNAse, whereas the free,uncomplexed DNA is not. It should be noted that at the optimaldispersion/DNA ration nearly all DNA were complexed with liposomes (notshown in FIG. 8).

EXAMPLE XXV Relationship Between Delivery Activity and Cytotoxicity ofthe Cationic Lipid Complex

This was studied by using a dispersion composed of compound XIV and DOPE(3:2, molar ratio). A431 human epidermoid carcinoma cells were used forthe transfection experiments. A fixed amount of DNA (4 μg) was mixedwith an increasing amount of cationic lipid dispersion or a commerciallyavailable transfection reagent, Lipofectin, and added to the A431 cellsfor transfection (FIG. 9). The toxicity of the treatment to the cellswas measured as the total amount of cellular protein extractable at thetime of CAT activity assay. As can be seen from the Figure, Lipofectintreated cells showed a greatly reduced protein content with 50%inhibition occurring at about 7 μg lipid/ml. Cells treated with thedispersion containing compound XIV and DOPE showed less toxicity; theIC₅₀ occurred at about 25 μg lipid/ml. The novel cationic cholesteroldispersion had also produced higher CAT activities than.Lipofectin.. Itis important to note that maximal CAT activity of cells treated withLipofectin occurred at the Lipofectin concentration of 15 μg/ml. At thisconcentration only about 12% of the total cellular proteins could berecovered from the culture. On the other hand, maximal CAT activity ofcells treated with the cationic cholesterol dispersion occurred at 20μg/ml; about 80% of the total cellular protein still remained in theculture at this concentration. Thus, the novel cationic cholesteroldispersion is more potent in the delivery activity and is also lesstoxic to the treated cells.

EXAMPLE XXVI Stability of the Cationic Cholesterol Derivatives

Lipid dispersions were prepared with various cationic cholesterolderivatives and DOPE (about 1:1 molar ratio). The transfectionactivities of the dispersions were tested at different times after thedispersions were stored at 4° C. in PBS, pH 7.5. Of the derivativeslisted in Table 1, only the dispersions containing compounds XIV and XVwere stable after storage; their transfection activities did not changefor at least 2 months. On the other hand, the dispersions composed ofother derivatives lose activity after 2-3 days in storage. Compounds XIVand XV contain a carbamoyl linker bond whereas other compounds containeither an ester bond or an amide bond. It is known that ester and amidebonds are more sensitive than the carbamoyl bond to hydrolysisparticularly in the presence of bases. The cationic derivatives maycatalyze the hydrolysis of each other's ester bonds, leading to theinactivation of the delivery activity. Compounds containing carbamoyllinker bonds are less sensitive to the base-catalyzed hydrolysis, yetthey can still be hydrolysed by cellular enzymes, i.e., they arebiodegradable. This is in contrast to the non-degradable ether bond inDOTMA which is the active ingredient of Lipofectin. Thus, a carbamoylbond seems to be the best choice for the linker bond of the cationiclipids as a delivery vehicle for polyanions.

Various of the features of the invention which are believed to be neware set forth in the appended claims.

We claim:
 1. A substantially non-toxic biodegradable amine derivative ofcholesterol having a cholesteryl group, a —NH—(CO)—O—* linker, whichlinker is linked by the side marked by the asterisk to the 3 position ofthe cholesteryl group and by the other side via the nitrogen to aspacer, the spacer having 1 to 15 carbon atoms in a branched or linearhydrocarbon, which is linked on its other side to a tertiary aminogroup.
 2. A derivative of cholesterol of claim 1 wherein the spacer is—(CH₂)₂—.
 3. A cationic lipid of the derivative of cholesterol ofclaim
 1. 4. A dry lipid film of the derivative of cholesterol of claim3.
 5. A hydrated lipid film of the derivative of cholesterol of claim 4.6. A substantially non-toxic biodegradable amine derivative ofcholesterol which is3β[N-(N¹-N¹-dimethylaminoethane)carbamoyl]cholesterol.
 7. A cationiclipid of the derivative of cholesterol of claim
 6. 8. A crystallineproduct of the derivative of cholesterol of claim
 6. 9. A dry lipid filmof the derivative of cholesterol of claim
 6. 10. A hydrated lipid filmof the derivative of cholesterol of claim
 9. 11. A substantiallynon-toxic biodegradable derivative of cholesterol which is3β[N-(polyethyleneimine)-carbamoyl]cholesterol.
 12. A cationic lipid ofthe derivative of cholesterol of claim
 11. 13. A lyophilate of thederivative of cholesterol of claim
 9. 14. A substantially non-toxicbiodegradable amine derivative of cholesterol having a cholesterylgroup, a linker selected from the group consisting of a —NH—(CO)—* and a—O—(CO)—* (reverse ester) linker, which linker is linked by the sidemarked by the asterisk to the 3 position of the cholesteryl group and bythe other side via the nitrogen or the oxygen, respectively, to abranched or linear hydrocarbon spacer having 1 to 15 carbon atoms, whichis linked on its other side to an amino group selected from the groupconsisting of a primary, tertiary or quaternary groups.
 15. A derivativeof cholesterol of claim 14, wherein the spacer is of 2 to 3 carbonatoms.
 16. A derivative of cholesterol of claim 15 wherein the spacer is—(CH₂)₂—.
 17. A substantially non-toxic biodegradable amine derivativeof cholesterol of claim 14 wherein the linker is a —NH—(CO)—* linker,and the amino group is primary.
 18. A derivative of cholesterol of claim17 which is cholesteryl-3β-carboxyamidoethylamine.
 19. A substantiallynon-toxic biodegradable amine derivative of cholesterol of claim 16,wherein the linker is a —NH—(CO)—* linker and the amino group areselected from the group consisting of one tertiary and one quaternaryamino group.
 20. A derivative of cholesterol of claim 19 wherein thederivative of cholesterol is selected from the group consisting ofcholesteryl-3β-carboxyamidoethylenedimethylamine andcholesteryl-3β-carboxyamidothylenetrimethlammonium iodide.
 21. A powderor a lyophilate of a derivative of cholesterol of claim
 20. 22. Aderivative of cholesterol of claim 20 which ischolesteryl-3β-carboxyamidoethylenedimethylamine.
 23. A derivative ofcholesterol of claim 20 which ischolesteryl-3β-carboxyamidoethylene-trimethylammonium iodide.
 24. Asubstantially non-toxic biodegradable amine of the derivative ofcholesterol of claim 16 wherein the linker is —O—(CO)—*, and the aminogroup is selected from the group consisting of one tertiary and onequaternary amino group.
 25. A derivative of cholesterol of claim 24wherein the derivative of cholesterol is selected from the groupconsisting of 1,3-bis-dimethylamino-2-propyl-cholesteryl-3β-carboxylateand 1-dimethylamino-3-trimethylamino-DL-2-propyl cholesteryl carboxylateiodide.
 26. A derivative of cholesterol of claim 25, which is1,3-bis-dimethylamino-2-propyl-cholesteryl-3β-carboxylate.
 27. Asubstantially non-toxic biodegradable amine derivative of cholesterolhaving a cholesteryl group, a —(CO)—O—* ester linker which a linker islinked by the side marked by an asterisk to the 3 position of thecholesteryl group and by the other side via the carbon to a 5 to 8carbon atoms branched or linear heteroatom spacer comprising in additionto hydrocarbons, at least one atom selected from the group consisting ofoxygen and nitrogen, which spacer is linked on its other side to anamino group consisting of one or two tertiary amino groups or one or twoamino quaternary groups.
 28. A derivative of cholesterol of claim 26wherein the spacer has a nitrogen and an oxygen atom.
 29. A derivativeof cholesterol of claim 27 which ischolesteryl-3β-oxysuccinamidoethylenedimethylamine.
 30. A derivative ofcholesterol of claim 28 which ischolesteryl-3β-oxysuccinamidoethylenetrimethylammonium iodide.